Many diseases are associated with an abnormal proliferation of blood vessels. The process of forming new blood vessels is termed angiogenesis. Under normal or non-pathologic conditions angiogenesis occurs under well-defined conditions such as in wound healing, in response to ischemia and during embryonal and fetal development. However, persistent or uncontrolled angiogenesis can lead to a variety of disease states or conditions and, in the case of solid tumors, may be a necessary condition to maintain the disease state. For example, angiogenesis occurs with neoplastic diseases, particularly with solid tumors, in autoimmune diseases, in collagenous vascular diseases such as rheumatoid arthritis, and in certain ophthalmalogical conditions such as diabetic retinopathy, retrolental fibroplasia and neovascular glaucoma. One therapeutic approach for the treatment of such diseases would be to restrict, reduce or eliminate the blood supply to the diseased cells or tissues. For example, solid tumors greater than a few millimeters undergo neovascularization without which further tumor growth would be impossible, so that inhibiting blood vessel formation will limit tumor size.
Some treatment strategies have attempted to limit the tumor's blood supply by occluding blood vessels supplying the tumor. For such treatment, the site of the tumor must be known and the tumor must be accessible. Thus a method of treatment that did not rely on knowing the location of or the accessibility to the site of interest would be valuable and could permit systemic delivery of a therapeutic anti-angiogenesis agent capable of specifically targeting a disease site.
Because of the role that angiogenesis plays in the development of disease, there is substantial interest in the development of angiogenesis inhibitors, especially where current therapies are less than optimal. Since endothelial cells are an integral part of blood vessel formation, a specific inhibitor of such cells would be advantageous in inhibiting angiogenesis, provided, of course, there is a minimum toxicity associated with that inhibitor. One particular target of interest is the endothelial cell-specific cadherin, VE-cadherin, that forms intercellular adherens junctions.
Cadherins are a family of cell adhesion molecules that are involved in the formation of specific cell-cell contacts (Takeichi, Ann. Rev. Biochem. 59: 237-252 (1990); Geiger & Ayalon, Ann. Rev. Cell Biol. 8: 302-332 (1992); Uemura, Cell 93: 1095-1098 (1998)). A number of members have been identified or characterized. Cadherins are single chain transmembrane glycoproteins with molecular weights of 120-140 kD. Members of this family exhibit calcium-dependent homophilic interactions and are responsible for the selective cell-cell recognition and adhesion, which is necessary for allocating different cell types to their proper places during organ development. Cadherins also play an important role in maintaining the integrity of multicellular structures. During embryonic morphogenesis the expression of diverse members of the cadherin family is spatially and temporally regulated facilitating the orderly assembly of various cell types into functional structures (Takeichi, Ann. Rev. Biochem. 59: 237-252 (1990)).
Members of the cadherins family have typical structural features and share considerable sequence homology (43-58%). Their extracellular region typically contains 5 repeating domains of approximately 110 amino acids. The N-terminal domain has been shown to be important in homotypic cell-cell interaction as evidenced by experiments with molecular chimeras, monoclonal antibodies and peptide inhibitors (Nose et al., Cell 54: 993-1001 (1988)). The 3-dimensional structures of the N-terminal domains of N-cadherin and E-cadherin have been elucidated (Shapiro et al., Nature 374: 327-337 (1995); Overduin et al., Science 267: 386-389 (1995); Nagar et al., Nature 380: 360-364 (1996)). Accordingly, it is believed that cadherins form dimers supported by zipper-like elements and possibly by disulfide linkage. The short intracellular portion of cadherins is their most highly conserved region and plays an essential role in classic cadherin function by anchoring cadherins to the cytoskeleton and providing signaling functions through cadherin phosphorylation (See, FIG. 1).
VE-cadherin (or cadherin-5) has been shown to be localized at intercellular junctions (adherens junctions) in cell-to-cell contacts (Lampugnani et al., J. Cell. Biol. 118: 1511-1522 (1992); Breviario et al., Arterioscler. Thromb. Vasc. Biol. 15: 1229-1239 (1995); Breier et al., Blood 87: 630-641 (1996); Lampugnani et al., J. Cell Biol. 129: 203-217 (1995)). A number of experimental observations suggest that this cadherin is involved in various aspects of vascular biology related to angiogenesis, including the assembly of endothelial cells into tubular structures (Bach et al., Experimental Cell Research 238: 324-334 (1998)). For example, thrombin-induced vascular permeability is shown to be associated with disassembly of endothelial adherens junctions (Rabiet et al., Arterioscler. Thromb. Vasc. Biol. 16: 488-496 (1996); Dejana, J. Clin Invest. 100: S7-10. (1997); Dejana et al., FASEB J., 9: 910-918 (1995); Dejana et al., Ann N Y Acad Sci. 811: 36-43 (1997); Gotsch et al., J. Cell. Sci. 110: 583-588 (1997); Kevil et al., J. Biol. Chem. 273: 15099-15103 (1998); Corada et al., Proc. Natl. Acad. Sci. 96: 9815-9820 (1999)). VE-cadherin and its N-terminal fragment inhibit the density-dependent growth (Yap et al., J. Cell Biol. 141: 779-789 (1998); Caveda et al., J. Clin. Invest. 98: 886-893 (1996)) and migration (Breviario et al., Arterioscler. Thromb. Vasc. Biol. 15: 1229-1239 (1995)) of endothelial cells. In other experiments, VE-cadherin was shown to confer adhesive properties to transfected cells (Breviario et al., Arterioscler. Thromb. Vasc. Biol. 15: 1229-1239 (1995); Breier et al., Blood 87: 630-641 (1996); Ali et al., Microcirculation 4: 267-277 (1997)), and an essential role for VE-cadherin in blood vessel formation has been demonstrated in VE-cadherin null mice. In these mice, severely impaired assembly of vascular structures leads to an embryonic lethal phenotype (Vittet et al., Proc. Natl. Acad. Sci. 94: 6273-6278 (1997); Faure et al., Development 128: 2093-2102 (1999); Carmeliet et al., Cell 98: 147-157 (1999)). These findings strongly validate VE-cadherin as an attractive pharmacological target for inhibiting neovascularization.
Prior to the present invention, attempts to use VE-cadherin antibody antagonists to prevent angiogenesis have been limited by the toxicity of the antibody to normal vasculature. For example, administering certain anti-cadherin antibodies in amounts sufficient to prevent or inhibit angiogenesis have resulted in disturbances in the integrity of normal vasculature with resultant vascular leak syndromes, hemorrhage and death. For example, the anti-VE -cadherin antibody 19E6 results in increased pulmonary vascular permeability because that antibody disrupts existing VE-cadherin-mediated cellular junctions as well as preventing formation of new VE-cadherin-mediated cellular adherens junctions. The present invention addresses now provides improved VE-cadherin antibody antagonists directed to particular sites on VE-cadherin and which overcome such problems.